Microscopic pK(a) values of Escherichia coli thioredoxin

Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pK(a) values of Trx have led to conflicting interpretations. Here,C-13-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pK(a) values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 ((C) under bar XXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pK(a) near 7.5 and has a well-defined, single-pK(a) titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pK(a) near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pK(a) near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXX (C) under bar) pK(a) of <11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.